All chemicals, if not specifically described, were from Sigma-Aldrich (St. Louis, MO, USA) or EMD Millipore, and all enzymes, unless otherwise noted, were obtained from New England Biolabs (Ipswich, MA, USA). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was obtained from TCI America (Portland, OR, USA) and [γ-32P]ATP was purchased from Perkin Elmer (Piscataway, NJ, USA). All unmodified oligodeoxyribonucleotides (ODNs) were from Integrated DNA Technologies (IDT, Coralville, IA, USA). The 12-mer ODNs harboring a site-specifically incorporated O4-alkyldT were synthesized using conventional phosphoramidite chemistry, as described previously (14 (link)). The identified and purities of all the lesion-harboring ODNs were confirmed by liquid chromatography-mass spectrometry (LC-MS) and tandem MS (MS/MS) analyses prior to their insertion into double-stranded plasmids.
Unmodified oligodeoxyribonucleotides odns
Manufactured by Integrated DNA Technologies
Sourced in United States
Unmodified oligodeoxyribonucleotides (ODNs) are short, synthetic, single-stranded DNA molecules. They are designed to serve as building blocks for various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
γ 32p atp, by PerkinElmer (8 mentions)
1 1 1 3 3 3 hexafluoro 2 propanol hfip, by Tokyo Chemical Industry (4 mentions)
Shrimp alkaline phosphatase, by Thermo Fisher Scientific (3 mentions)
γ 32 link p atp, by PerkinElmer (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by American Type Culture Collection (1 mentions)
Streptomycin, by American Type Culture Collection (1 mentions)
10 protocols using unmodified oligodeoxyribonucleotides odns
1
Characterization of O4-alkyldT Lesions
2
Polymerase-mediated DNA Synthesis Assay
3
Synthesis and Characterization of 5hmU-containing Oligonucleotide
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Unmodified oligodeoxyribonucleotides (ODNs) used in this study were purchased from Integrated DNA Technologies (Coralville, IA, USA). [γ-32P]ATP was obtained from Perkin Elmer (Piscataway, NJ, USA). Shrimp alkaline phosphatase was obtained from the USB Corporation (Cleveland, OH, USA). T4 phage β-glucosyltransferase (T4 β-GT) and all other enzymes unless otherwise specified were purchased from New England BioLabs (NEB). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was purchased from TCI America (Portland, OR, USA). Chemicals unless otherwise noted were obtained from Sigma-Aldrich (St. Louis, MO, USA).
The 5hmU-containing ODN (5′-ATGGCG5hmU GCTAT-3′) was synthesized following previously published procedures [17] (link), and the identity of the modified ODN was confirmed by electrospray ionization-mass spectrometry (ESI-MS) and tandem MS (MS/MS) analyses [18] (link). We chose this particular sequence context because we previously conducted replication studies for a number of DNA lesions in the same sequence context [19] (link)–[21] (link).
The 5hmU-containing ODN (5′-ATGGCG
4
Synthesis and Characterization of Modified Oligonucleotides
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Unmodified oligodeoxyribonucleotides (ODNs)
used in this study were purchased from Integrated DNA Technologies
(Coralville, IA). [γ-32P]ATP was obtained from PerkinElmer
(Piscataway, NJ). Shrimp alkaline phosphatase (SAP) was obtained from
USB Corporation (Cleveland, OH). All other enzymes unless otherwise
specified were purchased from New England BioLabs (Ipswich, MA). 1,1,1,3,3,3-Hexafluoro-2-propanol
(HFIP) was purchased from TCI America (Portland, OR). Chemicals unless
otherwise noted were obtained from Sigma-Aldrich (St. Louis, MO).
The HEK293T human embryonic kidney epithelial cells were purchased
from ATCC (Manassas, VA). The siRNAs used in this study were purchased
from Thermo Dharmacon: human-TDG SMARTpool (L-003780)
and siRNA Control Non-Targeting pool (D-001210).
Modified cytosine-containing
ODNs (5′-ATGGCGX GCTAT-3′, X = 5mdC,
5hmdC, 5fdC, or 5cadC) were synthesized previously. The HPLC traces
for monitoring the purities of these ODNs are shown in Figure S1 (Supporting Information ), and the identities of
these ODNs were confirmed by electrospray ionization–mass spectrometry
(ESI-MS) and tandem MS (MS/MS) analyses.22 (link)
used in this study were purchased from Integrated DNA Technologies
(Coralville, IA). [γ-32P]ATP was obtained from PerkinElmer
(Piscataway, NJ). Shrimp alkaline phosphatase (SAP) was obtained from
USB Corporation (Cleveland, OH). All other enzymes unless otherwise
specified were purchased from New England BioLabs (Ipswich, MA). 1,1,1,3,3,3-Hexafluoro-2-propanol
(HFIP) was purchased from TCI America (Portland, OR). Chemicals unless
otherwise noted were obtained from Sigma-Aldrich (St. Louis, MO).
The HEK293T human embryonic kidney epithelial cells were purchased
from ATCC (Manassas, VA). The siRNAs used in this study were purchased
from Thermo Dharmacon: human-TDG SMARTpool (L-003780)
and siRNA Control Non-Targeting pool (D-001210).
Modified cytosine-containing
ODNs (5′-ATGGCG
5hmdC, 5fdC, or 5cadC) were synthesized previously. The HPLC traces
for monitoring the purities of these ODNs are shown in Figure S1 (
these ODNs were confirmed by electrospray ionization–mass spectrometry
(ESI-MS) and tandem MS (MS/MS) analyses.22 (link)
5
Oligonucleotide Synthesis and DNA Repair Strains
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All chemicals, unless otherwise specified, were purchased from Sigma-Aldrich (St Louis, MO, USA) or EMD Millipore (Billerica, MA, USA). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was obtained from Oakwood Products Inc. (West Columbia, SC, USA). Common reagents for solid-phase DNA synthesis were obtained from Glen Research Co. (Sterling, VA, USA) and unmodified oligodeoxyribonucleotides (ODNs) were from Integrated DNA Technologies (Coralville, IA, USA). [γ-32P]ATP was obtained from Perkin Elmer (Piscataway, NJ, USA). Shrimp alkaline phosphatase was purchased from USB Corporation (Cleveland, OH, USA) and all other enzymes were obtained from New England Biolabs (Ipswich, MA, USA).
M13mp7(L2), wild-type AB1157 and C215 E. coli strains, and Ada- and Ogt-deficient E. coli strains (as FC215 derivatives), including C216 (Δogt::kan), C217 (Δada ΔalkB::cam) and C218 (Δogt::kan Δada ΔalkB::cam), were kindly provided by Prof. John M. Essigmann (35 (link)). Polymerase-deficient AB1157 strains [Δpol B1::spec (Pol II-deficient), ΔdinB (Pol IV-deficient), ΔumuC::kan (Pol V-deficient) and Δpol B1::specΔdinB ΔumuC::kan(Pol II, Pol IV, Pol V-triple knockout)] were generously provided by Prof. Graham C. Walker (36 (link)).
M13mp7(L2), wild-type AB1157 and C215 E. coli strains, and Ada- and Ogt-deficient E. coli strains (as FC215 derivatives), including C216 (Δogt::kan), C217 (Δada ΔalkB::cam) and C218 (Δogt::kan Δada ΔalkB::cam), were kindly provided by Prof. John M. Essigmann (35 (link)). Polymerase-deficient AB1157 strains [Δpol B1::spec (Pol II-deficient), ΔdinB (Pol IV-deficient), ΔumuC::kan (Pol V-deficient) and Δpol B1::specΔdinB ΔumuC::kan(Pol II, Pol IV, Pol V-triple knockout)] were generously provided by Prof. Graham C. Walker (36 (link)).
6
Synthesizing O2-alkyldT-containing Oligonucleotides
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All chemicals, unless otherwise specified, were from Sigma-Aldrich (St. Louis, MO) or EMD Millipore. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was obtained from TCI America (Portland, OR). Shrimp alkaline phosphatase and [γ-32P]ATP were purchased from USB Corporation (Cleveland, OH) and Perkin Elmer (Piscataway, NJ), respectively, and all other enzymes were obtained from New England Biolabs (Ipswich, MA). All unmodified oligodeoxyribonucleotides (ODNs) were purchased from Integrated DNA Technologies (Coralville, IA). The 12-mer ODNs harboring a site-specifically incorporated O2-alkyldT were synthesized using conventional phosphoramidite chemistry, as detailed below.
M13mp7(L2) plasmid and wild-type AB1157 E. coli strains were kindly provided by Prof. John M. Essigmann, and polymerase-deficient AB1157 strains [Δpol B1::spec (Pol II-deficient), ΔdinB (Pol IV-deficient), ΔumuC::kan (Pol V-deficient), and Δpol B1::spec ΔdinB ΔumuC::kan (Pol II, Pol IV, Pol V-triple knockout)] were generously provided by Prof. Graham C. Walker.
M13mp7(L2) plasmid and wild-type AB1157 E. coli strains were kindly provided by Prof. John M. Essigmann, and polymerase-deficient AB1157 strains [Δpol B1::spec (Pol II-deficient), ΔdinB (Pol IV-deficient), ΔumuC::kan (Pol V-deficient), and Δpol B1::spec ΔdinB ΔumuC::kan (Pol II, Pol IV, Pol V-triple knockout)] were generously provided by Prof. Graham C. Walker.
7
Oligodeoxyribonucleotides Synthesis and Cell Lines
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Unmodified oligodeoxyribonucleotides (ODNs) were purchased from Integrated DNA Technologies (Coralville, IA). [γ-32P]ATP was obtained from Perkin Elmer Life Sciences, enzymes were from New England Biolabs (NEB), and chemicals unless otherwise noted were from Sigma-Aldrich. The pSVRLuc plasmid backbone was kindly provided by Professor Orlando Schärer (21 (link)). HEK293T cells depleted of Pol η, Pol ι, Pol κ or Pol ζ were obtained by the CRISPR–Cas9 genome editing method, as described previously (22 (link)).
8
Investigating DNA Repair Mechanisms
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Unmodified oligodeoxyribonucleotides (ODNs) were purchased from Integrated DNA Technologies. [γ-32P]ATP was obtained from Perkin-Elmer. All enzymes and chemicals unless otherwise specified were obtained from New England BioLabs and Sigma-Aldrich, respectively. ON-TARGETplus SMARTpool siRNA against human TDG (L-003780) and Non-Targeting control siRNA (D-001210) were from Thermo Scientific Dharmacon. The 293T human embryonic kidney epithelial cells were purchased from ATCC. XPA-deficient (XP12RO) and XPA-complemented (GM15876A) human fibroblast cell lines were kindly provided by Prof. Karlene A Cimprich53 (link). Cells were cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin, and 100 µg/mL streptomycin (ATCC), and incubated at 37°C in 5% CO2 atmosphere.
9
Synthesis and Purification of Radio-labeled DNA
10
Polymerase Acquisition for Assays
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